In addition to normal decarboxylation of glutamate to 4-aminobutyrate, glutamate decarboxylase from pig brain was shown to catalyse decarboxylation-dependent transamination of L-glutamate and direct transamination of 4-aminobutyrate with pyridoxal 5'-phosphate to yield succinic semialdehyde and pyridoxamine 5'-phosphate in a 1:1 stoichiometric ratio. Both reactions result in conversion of holoenzyme into apoenzyme. With glutamate as substrate the rates of transamination differed markedly among the three forms of the enzyme (0.008, 0.012 and 0.029% of the rate of 4-aminobutyrate production by the alpha-, beta- and gamma-forms at pH 7.2) and accounted for the differences among the forms in rates of inactivation by glutamate and 4-aminobutyrate. Rates of transamination were maximal at about pH 8 and varied in parallel with the rate constants for inactivation from pH 6.5 to 8.0. Rates of transamination of glutamate and 4-aminobutyrate were similar, suggesting that the decarboxylation step is not entirely rate-limiting in the normal mechanism. The transamination was reversible, and apoenzyme could be reconstituted to holoenzyme by reverse transamination with succinic semialdehyde and pyridoxamine 5'-phosphate. As a major route of apoenzyme formation, the transamination reaction appears to be physiologically significant and could account for the high proportion of apoenzyme in brain.

Transaminations catalysed by brain glutamate decarboxylase.

Reaction Specificity of Native and Nicked 3,4-Dihydroxyphenylalanine Decarboxylase *

1.

The substrate-promoted inactivation of glutamate decarboxylase from hog brain was studied.




2.

Inactivation was a slow process that was dependent on the concentration of glutamate.




3.

Glutamate-dependent inactivation was not first order but was best described as the sum of two exponential decay processes. At 10 mM glutamate, the half-lives at 30°C were about 6 min for the fast component and 70 min for the slow component.




4.

Glutamate-dependent inactivation appeared to be due to the formation of apoenzyme since the rate and extent of inactivation were greatly reduced by the presence of pyridoxal 5′-phosphate (the cofactor, pyridoxal-P). Also, inactivated enzyme could be reactivated by adding pyridoxal-P (Meeley and Martin, 1983).




5.

Micromolar concentrations of ATP enhanced glutamate-promoted inactivation in the absence of pyridoxal-P. ATP also enhanced inactivation in the presence of 10µM pyridoxal-P, but somewhat higher concentrations were required for an equal effect. ATP had little or no direct effect on the enzyme in the absence of glutamate.




6.

In the absence of pyridoxal-P, Pi reduced the enhancement of inactivation by 10µM but not by 750µM ATP.




7.

Glutamate-promoted inactivation, its enhancement by ATP, and the opposition to inactivation by pyridoxal-P and Pi appear to be important in the regulation of glutamate decarboxylase.

E. Barboni

Papers

An abnormal reaction occurring in the presence of L-aromatic aminoacid decarboxylase.